The present invention relates to the general technology of plant genetic engineering and, in particular, to the identification of tissue-specific promoters and the use of these promoters to create novel genetically transformed plants.
Cereal grains, such as barley, wheat, rice, maize and oats, are the basic constituents of the food and feed industries. Because of their importance, considerable resources have been devoted toward the development of cereal grains having economically advantageous qualities. These qualities include increased vigor, disease resistance, greater yields, extended shelf-life, and enhanced nutritional content.
Unfortunately, cereal grains also serve as good hosts for a variety of fungal diseases. Several fungal diseases continue to cause heavy losses in both barley and wheat crops. One such disease is caused by the fungus Fusarium graminearum, which causes "scab" disease found on seed bearing spike tissues.
Aided by climatic conditions and the adoption of no-till agriculture, Fusarium graminearum, and its associated mycotoxins, has emerged as a major threat to food, feed and beverage products. Annually, billions of dollars worth of barley and wheat grains are lost to Fusarium, as crops infested with the fungus must be destroyed due to the lethality of grains containing more than two ppb of the Fusarium mycotoxin, "vomitoxin". At this time, there are no barley or wheat lines resistant to Fusarium and its associated fungal disease.
Ironically, barley seed already natively produces a protein, known as thionin, which inhibits Fusarium. However, in native barley, thionin is only present in sufficient quantities inside of the barley seed and not in the tissue surrounding the seed. Thus, the fungal infection can grow in the surrounding plant tissue in quantity and then infect the seed as well.
Modern techniques of recombinant DNA manipulation and genetic engineering offer the prospect of creating cereal lines which are resistant to fungal diseases. In recent years a number of genetic manipulations have been performed to modify various characteristics of cereal grains, often with commercially useful results. Such molecular approaches have definite advantages over classical breeding. The most obvious advantages are the highly directed nature of the genetic engineering process and the accelerated development of genetically engineered varieties with enhanced traits. That is, specific biochemical functionality provided by proteins produced by single genes or small related gene families, can be targeted for modification by overexpressing the particular gene involved in the target step, thereby "enhancing" or "accelerating" the targeted cellular process. Conversely, a biochemical process can be reduced or inhibited by inhibiting the expression of a gene or gene family. Approaches such as these can specifically modify a trait in an already existing, commercially useful plant without affecting other desired traits.
Gene expression is better altered by using tissue-specific promoters to target and restrict the desired genetic modifications to specific tissues in order to have a minimal effect on the overall growth and development of the plant. Promoters are DNA elements that direct the transcription of RNA in cells. Tissue-specific promoters help control the development of organisms together with other regulatory elements that determine tissue and temporal specificity of gene expression. This approach to genetic engineering, however, depends on the availability of a promoter specific enough to limit the expression of a particular transgene to the organ of interest, while simultaneously allowing expression at a high enough level to effectively modify the target gene in the desired tissues.
Presently, there are several promoters capable of expressing heterologous genes in a variety of plant tissues, but none capable of expressing solely in spike tissues. The promoters include barley B22E (Klemsdal, et al., "Primary Structure of a Novel Barley Gene Differentially Expressed in Immature Aleurone Layers," Molecular and General Genetics, 228:9-16 (1991)), which is expressed in aleurone cells, barley pZE40 (Smith, et al., "Temporal and Spatial Regulation of a Novel Gene in Barley Embryos," Plant Molecular Biology, 20:255-266 (1992), which is expressed in embryos, and a rice anther-specific gene, RTS2 (Lee and Hodges (1994), Genbank Accession: U12171; GI:607895). What is needed is a promoter that will allow the targeting of chimeric gene expression in the spike tissue of cereal grains.